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Characterization of brainstem preproglucagon projections to the paraventricular and dorsomedial hypothalamic nuclei
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a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m
w w w. e l s e v i e r. c o m / l o c a t e / b r a i n r e s
Research Report
Characterization of brainstem preproglucagon projections to the paraventricular and dorsomedial hypothalamic nuclei Niels Vrang a,b,⁎, Mikkel Hansen b , Philip Just Larsen a,b , Mads Tang-Christensen a,b a
Rheoscience, Glerupvej 2, 2610 Rødovre, Denmark Dept Anatomy, University of Copenhagen, Denmark
b
A R T I C LE I N FO
AB S T R A C T
Article history:
In the brain preproglucagon expression is limited to a cluster of neurons in the caudal part of
Accepted 7 February 2007
the nucleus of the solitary tract (NTS) as well as a smaller number of neurons that extend
Available online 27 February 2007
laterally from the NTS through the dorsal reticular area into the A1 area. These neurons process preproglucagon to glucagon-like peptide-1 (GLP-1), GLP-2, oxyntomodulin and
Keywords:
glicentin. The neurons project mainly to the hypothalamus, where especially two nuclei
Nucleus of the solitary tract
involved in appetite regulation – the paraventricular (PVN) and dorsomedial (DMH)
Preproglucagon
hypothalamic nuclei – are heavily endowed with GLP-immunoreactive nerve fibres. To
Appetite regulation
gain further insight into this neurocircuitry, we injected the retrograde tracers cholera toxin,
Paraventricular nucleus of
subunit B (ChB) and Fluorogold (FG) into the PVN and the DMH, respectively. Of thirty-five
the hypothalamus
injected rats, six had successful injections that predominantly restricted within the
Dorsomedial nucleus of
boundaries of the PVN and DMH. Hindbrain sections from these rats were triple labelled
the hypothalamus
for ChB, FG and GLP-2. A total of 24 ± 1% of the PVN-projecting NTS-neurons contained GLP2-ir whereas 67 ± 4% of the DMH-projecting neurons were also stained for GLP-2, suggesting that the NTS-projections to the DMH arise mainly from preproglucagon neurons. Approximately 20% of backfilled cells in the NTS contained both retrograde tracers, therefore presumably representing neurons projecting to both the PVN and the DMH. The results of the present study demonstrate that the majority of the preproglucagonexpressing neurons in the NTS project in a target-specific manner to the hypothalamus. It is therefore possible that individual subgroups of GLP-containing neurons can mediate different physiological responses. © 2007 Elsevier B.V. All rights reserved.
1.
Introduction
The nucleus tractus solitarius (NTS) receives visceral afferent information from the cardiovascular, respiratory, gastrointestinal and taste systems in which it plays an important role as both a relay and integrative center (Blessing, 1997). The NTS is topographically organised as different sensory modalities are received and processed at different rostro-caudal levels.
The complexity of the nucleus is highlighted by the numerous neurotransmitters synthesized by neurons in the NTS (Maley, 1996). Over the past decade, the function of a small population of preproglucagon-expressing neurons in the caudal “gastrointestinal” part of the NTS has been scrutinized. The processing of preproglucagon within these neurons is similar to the processing in intestinal L-cells giving rise to mainly glucagonlike peptide-1 (GLP-1) and -2 (GLP-2) and smaller amounts of
⁎ Corresponding author. Rheoscience, Glerupvej 2, 2610 Rødovre, Denmark. Fax: +45 4450 1962. E-mail address: [email protected] (N. Vrang). 0006-8993/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2007.02.043
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glicentin and oxyntomodulin (Bell et al., 1983; Holst et al., 1994; Larsen et al., 1997). Since preproglucagon expression in the brain is limited to the NTS, it has been possible to investigate the projections of these neurons by immunocytochemistry using antibodies raised against preproglucagon or preproglucagon-derived peptides (Jin et al., 1988; Larsen et al., 1997; Rinaman, 1999). These studies have collectively shown that the major target of the brainstem preproglucagon neurons is the hypothalamus. In the hypothalamus, the densest innervation has been observed in two nuclei implicated in the control of food intake and bodyweight, namely the paraventricular (PVN) and dorsomedial (DMH) hypothalamic nuclei. In line with the preproglucagon projection pattern intracerebroventricular (i.c.v.) administration of either GLP-1, GLP-2 or oxyntomodulin has been shown to inhibit food intake in rats (Dakin et al., 2001; Tang-Christensen et al., 1996, 1998, 2000; Turton et al., 1996). Whereas oxyntomodulin presumably acts via the GLP-1 receptor, GLP-2 specifically activates the GLP-2 receptor (Druce and Bloom, 2006; Munroe et al., 1999). Recent studies have revealed an intriguing complexity of the brainstem–hypothalamic preproglucagon system. Whereas the GLP-1 receptor mRNA is expressed in all hypothalamic areas receiving GLP-immunoreactive fibers (Merchenthaler et al., 1999), the GLP-2 receptor expression in the hypothalamus is confined to the compact part of the DMH (Tang-Christensen et al., 2000). The differential distribution of GLP-1 and GLP-2 receptors indeed suggest that GLP-1 and GLP-2 play different roles in appetite regulation despite the fact that the peptides are produced in the same neurons and are derived from the same precursor peptide. To better understand the anatomy of the brainstem–hypothalamic preproglucagon system we employed double retrograde tracing from the PVN and DMH in combination with GLP-2 immunohistochemistry. Using this approach we sought to topographically characterize preproglucagon projections and determine whether the preproglucagon neurons project in a site-specific fashion to the hypothalamus, or whether single GLP-containing neurons target multiple sites.
2.
Results
A total of six rats had injections that were centered in both the PVN and the DMH. The center of the ChB and FG injection sites are shown in Fig. 1. Animals with ChB injections spreading caudally in the DMH or with FG injections spreading rostrally in the PVN were not included in the analysis. As seen in Fig. 1, rats T2140, T2065 and T2114 had ChB injection sites that were centered in the caudal part of the PVN (Plate #27, Swanson, 1998) whereas rat numbers T2126, T2116 and T2138 were centered at the level of the posterior magnocellular PVN (Plate Fig. 1 – Location of ChB and Fluorogold injections in the rats selected for further analysis. A and B are medium power photomicrographs showing the size and location of the PVN (A) and DMH (B) injections (single stained using DAB as chromagen) from rat T2138. The extent and location of the remaining 5 rats have been plotted onto Adobe Illustrator maps from the rat atlas by Swanson (1998). In general, ChB injections are smaller and more clearly demarcated than FG injections (compare A and B). Scale bars in A, B = 100 μm.
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#26, Swanson, 1998). Only animals with FG injections involving the ventral subnucleus of the DMH – that receives the densest GLP-ergic innervation (Larsen et al., 1997) – were included in the analysis. Animals T2140, T2126, T2114 and T2116 were centered in the mid-DMH (Plate # 30, Swanson, 1998), the remaining (T2065, T2138) at a slightly more caudal level (Plate
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#31, Swanson, 1998). In the present study we used a monoclonal mouse antibody raised against human GLP-2 and shown to give rise to the same pattern of immunoreactivity as a previously characterized polyclonal GLP-1 antibody (Larsen et al., 1997). The availability of a monoclonal antibody facilitated the visualization of ChB, FG and GLP-2 in the same sections. Both ChB injections into the PVN and Fluorogold injections into the DMH gave rise to retrogradely labeled cells in the nucleus of the solitary tract. An example of triple stained neuron in the NTS is shown in Fig. 2 and a map of single-, double- and triple-labeled cells in one representative rat is shown in Fig. 3. Cell counts revealed that approximately 25% of ChBlabeled cells in the NTS co-stored GLP-2, whereas 67% of the FG cells co-stored GLP-2, suggesting that the majority of the projection from the NTS to the DMH contain preproglucagonderived peptides. Since preproglucagon-expressing neurons are also found in the reticular formation extending laterally and ventrally from the NTS-proper, cell counts were broken down by region (NTS-proper and “lateral extension”). This part
of the reticular formation contained neurons retrogradely labeled with both ChB and FG and the degree of co-localization with GLP-2-ir was almost identical to that found in the NTSproper (see Table 2). Cell counts were also broken down by rostro-caudal level of the NTS (caudal to the AP, level of the AP and rostral to the AP). These counts revealed that the majority of NTS neurons projecting to the PVN (ChB-labeled) were found at the level of the AP, whereas the majority of the DMHprojection neurons were found in the caudal part of the NTS (see Table 2). As a reflection of the fact that the highest number of GLP-2-positive neurons was found in the caudal part of the visceral NTS (caudal to the AP), this region also contained the highest degree of co-localization between ChB and GLP-2 (45%) and FG and GLP-2 (82%). Furthermore the counting revealed (see Table 1) co-localization between ChB and FG (=neurons projecting to both the PVN and the DMH). Approximately three-fourths of these “two-target” neurons were also GLP-2-positive.
3.
Discussion
This study shows that preproglucagon projections constitute the predominant input from the nucleus of the solitary tract to the dorsomedial hypothalamic nucleus. While approximately 65% of NTS-neurons projecting to the DMH co-stored the preproglucagon-derived peptide GLP-2 only 25% of the NTSneurons projecting to the PVN were found to be GLP-ergic. Furthermore, the study revealed that the majority of the NTSneurons project to either the PVN or the DMH. Only 15–25% of the retrogradely labeled cells were found to project to both hypothalamic nuclei.
3.1.
Fig. 2 – Fluorescence micrographs showing examples of single, double and triple labeled cells in the NTS. A and B are images of the same area but viewed with different emission/ excitation filters. Thin arrow in A and B point to a single neuron labeled with both ChB (red in A), Fluorogold (yellow granula in B) and GLP-2 (blue in B). Large arrow in B points to a neuron containing both FG granula and GLP-2 immunoreactivity. Additionally a couple of single-labeled GLP-2 neurons are seen (arrowheads). Scale bars = 50 μm.
Technical considerations
Clearly, because of the inherent inter-individual differences when performing in vivo neuronal tracing, double-retrograde tracing warrants careful interpretation of the results. In the present study, the two sensitive and well-characterized retrograde tracers cholera toxin, subunit B (ChB) and Fluorogold were used (Lennard et al., 1993; Luppi et al., 1990; Merchenthaler, 1991; Schmued and Fallon, 1986). Because the preproglucagon projections target the PVN and DMH-proper rather than the surroundings, confining the injections to the individual nuclei was not a primary concern, although injections into the DMH extending rostrally into the PVN were excluded from further analysis. Since the GLP-containing nerve fibers course through the periventricular strata on the way to the PVN (Larsen et al., 1997), injections close to the third ventricle at the level of the DMH could potentially damage passing fibers, leading to uptake and false-positive doublelabeled cells. Although the DMH injections inevitably spread into the periventricular strata no injections were centered here (see Fig. 1), making this possibility less likely.
3.2.
Anatomical considerations
A number of studies have examined the projections from the NTS to the hypothalamus using both anterograde and retrograde tracing techniques (Ricardo and Koh, 1978; Sawchenko
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Fig. 3 – Single-, double- and triple-labeled ChB, FG and GLP-2 containing neurons are plotted in single 40-μm tissue sections through the caudal NTS. The distribution of cells in two rats with successful PVN and DMH injections of ChB and Fluorogold, respectively, is shown. The labeling is as follows: GLP-2 (red dots), ChB (open squares), FG (open stars), ChB/GLP-2 co-loc (red squares), FG/GLP-2 co-loc (red stars), ChB/FG co-loc (black triangles), ChB/FG/GLP-2 co-loc (red triangles). The drawings are arranged from rostral (top) to caudal (bottom) and are redrawn from the atlas by Swanson (1998, plates 70 to 73).
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Table 1 – Total and individual cell counts and degree of co-localization between the three antigens examined (cell counts performed in entire visceral NTS and includes the cells extending from the lateral NTS into the RVLM)
# ChB cells # FG cells # GLP-2-ir cells % ChB with GLP-2 % FG with GLP-2 % ChB with FG % FG with ChB % FG/ChB with GLP-2
Total
T2065
T2114
T2116
T2126
T2138
T2140
65 ± 5 45 ± 5 141 ± 17 24 ± 1 67 ± 4 15 ± 1 23 ± 3 72 ± 7
40 39 191 20 72 13 13 100
55 67 173 24 58 22 18 75
68 33 152 21 79 13 27 67
81 38 146 27 68 14 29 64
63 53 89 25 55 16 19 50
86 37 92 28 70 14 32 75
Note that counts were only made in one series of sections out of a total of six. Total counts are expressed as mean ± SEM of the 6 rats included in the study.
and Swanson, 1982; Ter Horst et al., 1989; Thompson and Swanson, 1998). Especially the brainstem projections to the PVN have been extensively scrutinized (Swanson, 1987). Catecholaminergic projections to the PVN have been demonstrated to originate not only in the NTS (A2/C2 region), but also the A1/C1 (caudal ventrolateral medulla) and A6 (locus coeruleus) regions contribute with catecholaminergic fibers to the PVN (Sawchenko and Swanson, 1982). A number of other neurotransmitters found in nerve terminals in the PVN, however, are found in the NTS. Aside from the GLP-1/2containing neurons (Larsen et al., 1997; Rinaman, 1999) also somatostatin-immunoreactive neurons (Sawchenko et al., 1990), cocaine- and amphetamine-regulated transcript (CART) (Fekete et al., 2004) and proopiomelanocortin (POMC) (Kiss et al., 1984) containing neurons in the NTS have been shown to project to the PVN. Our finding that approximately 25% of the neurons projecting to the PVN contain preproglucagon-derived peptides is in line with the complex neurochemistry of this projection. Much less is known about the neurochemistry of NTS projections to the DMH as only a single study has addressed this subject (Sawchenko and Swanson, 1982). In a comprehensive paper on brainstem noradrenergic (and adrenergic) projections to the PVN and SON, the authors note that “in experiments with injections centered still more caudally, in
the dorsomedial nucleus proper, a similar pattern of singleand double-labeling was observed” (Sawchenko and Swanson, 1982). The authors found dopamine-beta-hydroxylase immunoreactivity in approximately 75–80% of true-blue containing neurons in the NTS (A2–C2 region) following PVN injections, and apparently (although not quantified) the same pattern was observed in cases accidentally involving the DMH (Sawchenko and Swanson, 1982). Although we have not examined the degree of co-localization between Fluorogold and catecholamine neuron markers our finding of a high degree of co-localization (approximately 65%) between Fluorogold and GLP-2-ir, suggests that catecholaminergic input from the NTS to the DMH constitute less than 35% of this connection, since the preproglucagon neuronal population is separate from the catecholaminergic neuronal pool in the C2/ A2 region (Larsen et al., 1997). In the present study, GLP-2-positive cells projecting to the PVN and DMH were found in both the NTS proper as well as in the wing of GLP-2-positive neurons extending laterally through the dorsal reticular area into the A1 area (at the level of the caudal part of the area postrema). The fact that similar degrees of co-localization were found in the NTSproper and the “lateral extension” indeed suggests that the pool of GLP-2 within and outside the NTS constitutes a functional homogeneous cell group rather than two separate
Table 2 – Cell counts and degree of co-localization divided into different anatomical sub regions
# ChB cells # FG cells # GLP-2-ir cells % ChB with GLP-2 % FG with GLP-2 % ChB with FG % FG with ChB % FG/ChB with GLP-2 # ChB with GLP-2 # FG with GLP-2 # ChB with FG # FG/ChB with GLP-2
Total
NTS proper
Lateral extension
Caudal NTS
NTS-AP-level
NTS-rostral to AP
65 ± 5 45 ± 5 141 ± 17 24 ± 1 67 ± 4 15 ± 1 23 ± 3 72 ± 7 16 ± 2 29 ± 2 10 ± 1 7±1
48 ± 7 34 ± 4 106 ± 11 25 ± 2 66 ± 5 16 ± 3 22 ± 4 66 ± 11 12 ± 1 21 ± 1 7±1 5±1
18 ± 6 11 ± 3 34 ± 8 24 ± 4 82 ± 9 21 ± 10 46 ± 18 72 ± 16 5±1 8±1 3±1 2±1
27 ± 3 28 ± 3 114 ± 17 45 ± 5 84 ± 4 25 ± 3 25 ± 3 79 ± 7 12 ± 1 23 ± 3 7±1 6±1
31 ± 6 14 ± 3 27 ± 5 11 ± 3 50 ± 12 8±3 25 ± 7 58 ± 19 4±3 6±2 3±1 2±1
10 ± 2 3±1 0 0 0 0 0 0 0 0 0 0
Number of labeled cells counted in the entire visceral NTS (total numbers include counts at all rostro-caudal levels and include cells located in the lateral extension of the NTS). These cell counts have been broken down into: 1) NTS-proper and the lateral extension of the NTS and 2) different rostrocaudal levels (caudal to the AP, at the level of the AP and rostral to the AP). Note that counts were only made in one series of sections out of a total of six. Total counts are expressed as mean ± SEM of the 6 rats included in the study.
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populations of neurons as would be predicted from topographic criteria. This is in line with previous data from our laboratory showing c-Fos induction following gastric distension in GLP-2-immunopositive neurons located both in the NTS proper and in the ventrolateral medulla (Vrang et al., 2003).
3.3.
Fig. 4 – Laser confocal images (5 μm thick optical sections) showing (A) GLP-1 and (B) GLP-2 immunoreactivity in the NTS. GLP-1-ir was visualized using a rabbit–polyclonal antibody (#2135) whereas GLP-2-ir was visualized using a mouse monoclonal antibody (#F1221). As seen in C the two peptides co-localize 100% in both neuronal and axonal-like structures. Scale bars = 50 μm.
Functional considerations
A number of studies have examined the possible functional role of the brainstem preproglucagon-expressing neurons. Based on the anatomy of the central preproglucagon system, we and others hypothesized a possible role of preproglucagonderived peptides in appetite regulation. Since the initial studies reporting anorectic effects of centrally administered GLP-1 (Tang-Christensen et al., 1996; Turton et al., 1996), a number of studies have demonstrated that indeed GLP-1 reduces food intake — an effect that is mediated by the GLP-1 receptor as GLP-1 receptor antagonists can block the effects (see Larsen et al., 2003). In contrast to the wide hypothalamic and brainstem distribution of GLP-1 receptors, the GLP-2 receptor expression is much more limited. In the hypothalamus the expression is limited to the DMH, but also few extra hypothalamic sites contain GLP-2 mRNA albeit at lower levels (hippocampus, premamillary nucleus, substantia nigra, lateral parabrachial nucleus) (Lovshin et al., 2004). The short-lasting anorectic effects of centrally administered GLP-2 is presumably mediated by the GLP-2 receptor-bearing neurons in the DMH, which was also the only hypothalamic area that contained a significantly higher number of c-Fos-ir cells following GLP-2 administration (Tang-Christensen et al., 2000). These data suggest that by activating DMH neurons a short-term reduction in food intake can take place. The DMH projects densely to parvocellular parts of the PVN that could be responsible for mediating the observed anorexia (Thompson et al., 1996). While it is generally accepted that the PVN plays a crucial role in regulating a variety of homeostatic functions (e.g. appetite, thirst, stress, temperature) the role played by the DMH in hypothalamic integration is somewhat controversial. A large number of lesion studies support the notion that the DMH plays a role in controlling the bodyweight set-point, in that animals with DMH lesions are capable of regulating their bodyweight, albeit at a lower level (Bernardis and Bellinger, 1998). Although interpretation of lesion studies is hampered by the complexity of the DMH, recently other data have accumulated that point to a role of the DMH in regulating energy balance. First, the DMH contains a population of PVNprojecting neuropeptide Y (NPY) mRNA-expressing neurons that are activated in lactating rats, i.e. at a time of increased body-energy demand (Li et al., 1998a,b). Second, NPY mRNA expression is increased in the DMH in the hyperphagic OLETF also pointing to a role of these neurons in animals with an increased drive to eat (Bi et al., 2001). Our present anatomical findings do not readily answer what differences might exist between GLP-1- and GLP-2mediated anorexia, but the fact that the preproglucagon neurons only have partly overlapping projection systems suggests that separate populations of preproglucagon neurons could be involved in different aspects of feeding behavior. The results presented here suggest that even within this relatively
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small group of neurons, separate populations with anatomically diverse projections exist highlighting the necessity of careful anatomical analyses as the basis to understand brain physiology.
4.
Experimental procedures
4.1.
Animals and surgery
Thirty-five male Wistar rats were used for the in vivo neuronal tracing experiments. The rats were kept on a 12-h light, 12-h dark cycle (lights on at 0600 h) in a temperature-controlled environment (22–24 °C) with free access to food (Altromin standard chow 1243; C. Petersen, Ringsted, Denmark) and water. All experiments were conducted in accordance with internationally accepted principles for the care and use of laboratory animals and were approved by the Danish Committee of Animal Research. Rats were deeply anaesthetised using hypnorm dormicum (1.0 ml/kg; containing 0.08 mg fentanyl, 2.5 mg fluanisone and 5.0 mg medazolam per ml) and placed in a Kopf stereotaxic apparatus. The incisor bar was set 2 mm below the ear bars. A glass electrode with a tip diameter less than 25 μm was filled with 4 μl of a cholera toxin, subunit B (ChB; choleragenoid, Sigma Chemicals Co., MO, USA) (1% w/v diluted in phosphate-buffered saline containing 50 mM potassium, KPBS) and another with Fluorogold (2% diluted in 0.9% NaCl). Both ChB and FG were injected iontophoretically using a positive current of 10 μA (7 s on, 7 s off) for 10 min, then left in situ for another 10 min to avoid spreading of tracer upon removal. Injection co-ordinates for ChB injections into the PVN were: antero-posterior = −1.8 mm (relative to bregma), lateral = 0.5 mm, and 7.6 mm below the dura. Injection co-ordinates for FG injections into the DMH were: antero-posterior = − 2.6 mm (relative to bregma), lateral = 0.5 mm, and 8.4 mm below the dura. At 7 days postoperatively, the animals were reanaesthetized and vascularly perfused with heparinized KPBS (15,000 IU/l), followed by 4% paraformaldehyde dissolved in 0.1 M phosphate buffer (pH = 7.4) for 15 min. The brains were removed and post-fixed in the same fixative overnight – then stored in KPBS until further processing. The brains were cryoprotected for two days in a 30% sucrose–KPBS solution and one-in-six series of 40-μm-thick frontal sections were cut (fore- and hindbrain separately) on a freezing microtome and collected in cryopreservant (Watson et al., 1986) and stored at −20 °C. All reactions were carried out on free-floating sections.
4.2.
Immunohistochemistry
4.2.1.
Single immunohistochemistry
To determine the precise injection sites one forebrain series from each rat was immunoreacted with an anti-FG antibody (rabbit anti-Fluorogold, Fluorochrome Inc., Englewood, CO) and one series with an anti-ChB antibody (goat anti-ChB; LIST Biologicals, Campbell, CA). Both antibodies were diluted 1:4000 and prepared in KPBS with 0.3% Triton X-100 and 1.0% bovine serum albumin (BSA). The sections were stained using the indirect avidin–biotin method and diaminobenzidine as a chromagen as described in detail previously (Vrang et al., 1995).
4.2.2. Double staining to determine the overlap of GLP-1 and GLP-2 (characterization of monoclonal GLP-2 antibody) While the monoclonal GLP-2 antibody used in the present study has been raised against full-length human GLP-2 (GLP21–33) and previously shown to stain neuronal cell bodies overlapping exactly with preproglucagon mRNA-expressing neurons in the NTS, the degree to which the GLP-2 immunoreactivity overlaps with GLP-1 immunoreactivity has not been examined (Tang-Christensen et al., 2000). In order to examine the degree of overlap between GLP-1-ir and GLP-2-ir in the brainstem, we used a double immunohistochemical detection procedure. GLP-1 immunoreactivity was detected using a GLP1 antibody (#2135; Larsen et al., 1997) and the double staining was performed according to the procedure below. As seen in Fig. 4, we found 100% co-localization between the two antibodies. These data support previous results demonstrating that preproglucagon in the NTS is processed to yield GLP-1, GLP-2 and oxyntomodulin (Larsen et al., 1997).
4.2.3.
GLP-1 and GLP-2 double staining procedure
Brainstem test sections from male Wistar rats perfused transcardially with 4% paraformaldehyde were reacted as follows: First, the sections were washed 3× for 10 min each in KPBS, then for 10 min in 0.1% H2O2 in KPBS followed by 20 min in 5% swine serum in KPBS with 0.3% TX and 1.0% BSA (KBPS– BT). After 3× for 10 min each in KPBS, the sections were incubated in the primary antibodies: mouse anti-GLP-2 diluted 1:200 (F12-21, generously provided by Jes T. Clausen, Novonordisk A/S; complete overlap with preproglucagon mRNA shown in Tang-Christensen et al., 2000) and rabbit anti-GLP-1 diluted 1:5000 (#2135, characterized in Larsen et al., 1997) in KPBS–BT. The next day, sections were rinsed 3× for 10 min each in KPBS containing 0.1% TX and 0.25% BSA (KPBS-T) then incubated in biotinylated donkey anti-rabbit 1:1000 (Jackson Immuno Research Laboratories) diluted in KPBS–BT. After 3 rinses in KPBS-T, 60 min in ABC–streptavidin horseradish peroxidase (Vector Elite Kit™; Vector Laboratories, Burlingame, CA) and 3 washes in KPBS-T, sections were reacted for 12 min in biotinylated tyramide (TSA; biotinylated according to the procedure described by Adams (1992) using tyramine–HCl (Sigma Aldrich) and sulfosuccinimidyl-6-(biotinimide) hexanoate (NHS–LC–biotin; Pierce, Rockford, IL)) diluted 1:100 in KPBS-T containing 0.005% H2O2. Sections were rinsed 3 times in KPBS-T incubated for 60 min in streptavidin Alexa 546 (Molecular Probes, Eugene, OR) diluted 1:200 in KPBS-T. Next, sections were rinsed 3× for 5 min each in KPBS-T and subsequently incubated for 10 min in 1% H2O2 in KPBS (30 ml 35% H2O2/ml) to block the added peroxidase. Following 3 rinses for 10 min each in KPBS-T, sections were incubated for 60 min in peroxidase-coupled secondary antibody (HRP-coupled antimouse antibody diluted 1:100 in KPBS-T; Jackson Immuno Research Laboratories). After 3 rinses in KPBS-T, sections were incubated for 12 min in fluorescein-labeled tyramine (FITC– TSA; labeled according to the procedure described by Adams (1992) using tyramine–HCl (Sigma Aldrich) and sulfosuccinimidyl-6-(fluorescein) hexanoate (NHS–LC–biotin; Pierce, Rockford, IL)). After this reaction, sections were rinsed 2× for 10 min each in KPBS-T and finally for 10 min in distilled water. Slides were dehydrated through a graded series of ethanol dilutions (30%, 60%, 80% 96% 99% and xylene) cover slipped in Pertex™.
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4.3.
Triple staining – GLP-2, ChB and FG co-localization
GLP-2, ChB and FG were visualized simultaneously by a double immunohistochemical detection procedure and direct visualization of FG using a UV-filter on the fluorescence microscope. Brainstem sections from rats with successful PVN and DMH injections were reacted as follows. Firstly, the sections were washed 3× for 10 min each in KPBS, then 10 min in 0.1% H2O2 in KPBS followed by a 20-min incubation in 5% swine serum in KPBS with 0.3% TX and 1.0% BSA. After 3 rinses for 10 min each in KPBS, the sections were incubated in the primary antibodies: mouse anti-GLP-2 diluted 1:6000 (F12-21, generously provided by Jes T. Clausen, Novonordisk A/S) and goat anti-ChB diluted 1:500 in KPBS–BT. The next day sections were rinsed 3× for 10 min each in KPBS–BT before incubation in biotinylated donkey anti-mouse 1:2000 (Jackson Immuno Research Laboratories). After 3 rinses for 10 min each in KPBST followed by 60 min in ABC–streptavidin horseradish peroxidase the sections were washed 3× for 10 min each in KPBS-T. Sections were then reacted for 12 min in biotinylated tyramide diluted 1:100 in KPBS-T containing 0.005% H2O2, rinsed 3× for 5 min each in KPBS-T and incubated for 60 min in Streptavidin Alexa 350 (Molecular Probes) diluted 1:200 in KPBS-T. Following 3 rinses for 5 min each in KPBS-T, the ChB antibody was detected by incubating the sections for 60 min with a Texas-Red-labeled anti-goat antibody diluted 1:100 in KPBS-T (Jackson Immuno Research Laboratories). After this reaction, sections were rinsed 2× for 10 min each in KPBS-T then dehydrated through a graded series of ethanol dilutions (30%, 60%, 80% 96% 99% and xylene) and cover slipped in Pertex™.
4.4.
Photomicrographs
Sections double-labeled with GLP-1 and GLP-2 were examined using a Zeiss LSM510 laser confocal microscope and images (5 μm thick optical sections) acquired using LSM510 software. Images of triple labeled (GLP-2, FG and CHB) cells were acquired using a Nikon ACT-1200 digital camera mounted on a Nikon E1000M microscope. Single, double and triple labeled cells were scored in one series of triplelabeled sections (brainstem cut in 6 series, hence sections were spaced 240 μm apart). All sections from this triplelabeled series through the NTS were scored for GLP-2, FG, ChB single-, double- and triple-labeled cells by an observer blinded to the different treatment groups. The AP was used to define three rostro-caudal levels of the visceral NTS: caudal to the AP, AP (sections containing the AP) and rostral to the AP. In addition the counts were divided into NTS and lateral extension of the NTS (lateral reticular formation that contains neurons projecting to the PVN and DMH as well as a group of GLP-positive neurons). Although GLP-2 and Fluorogold were visualized using the same filter settings, the grainy yellow Fluorogold contrasted clearly with the homogenous blue color emitted by the Alexa-350 dye (see Fig. 2). All images were enhanced for brightness and contrast in Adobe Photoshop 7.0 and combined into plates using Adobe Illustrator 10.0. Adobe Illustrator plates (Plates 70 to 73) from the atlas by Swanson (1998) were used to generate the map of labeled cells in Fig. 3.
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Acknowledgments This study was supported by The Danish Diabetes Association, The Novo Nordisk Foundation and Fonden til Lægevidenskabens Fremme. We thank Susi Jensen, Anja Daniel Andersen and Farida Sahebzadeh for excellent technical assistance.
REFERENCES
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a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m
w w w. e l s e v i e r. c o m / l o c a t e / b r a i n r e s
Research Report
Characterization of brainstem preproglucagon projections to the paraventricular and dorsomedial hypothalamic nuclei Niels Vrang a,b,⁎, Mikkel Hansen b , Philip Just Larsen a,b , Mads Tang-Christensen a,b a
Rheoscience, Glerupvej 2, 2610 Rødovre, Denmark Dept Anatomy, University of Copenhagen, Denmark
b
A R T I C LE I N FO
AB S T R A C T
Article history:
In the brain preproglucagon expression is limited to a cluster of neurons in the caudal part of
Accepted 7 February 2007
the nucleus of the solitary tract (NTS) as well as a smaller number of neurons that extend
Available online 27 February 2007
laterally from the NTS through the dorsal reticular area into the A1 area. These neurons process preproglucagon to glucagon-like peptide-1 (GLP-1), GLP-2, oxyntomodulin and
Keywords:
glicentin. The neurons project mainly to the hypothalamus, where especially two nuclei
Nucleus of the solitary tract
involved in appetite regulation – the paraventricular (PVN) and dorsomedial (DMH)
Preproglucagon
hypothalamic nuclei – are heavily endowed with GLP-immunoreactive nerve fibres. To
Appetite regulation
gain further insight into this neurocircuitry, we injected the retrograde tracers cholera toxin,
Paraventricular nucleus of
subunit B (ChB) and Fluorogold (FG) into the PVN and the DMH, respectively. Of thirty-five
the hypothalamus
injected rats, six had successful injections that predominantly restricted within the
Dorsomedial nucleus of
boundaries of the PVN and DMH. Hindbrain sections from these rats were triple labelled
the hypothalamus
for ChB, FG and GLP-2. A total of 24 ± 1% of the PVN-projecting NTS-neurons contained GLP2-ir whereas 67 ± 4% of the DMH-projecting neurons were also stained for GLP-2, suggesting that the NTS-projections to the DMH arise mainly from preproglucagon neurons. Approximately 20% of backfilled cells in the NTS contained both retrograde tracers, therefore presumably representing neurons projecting to both the PVN and the DMH. The results of the present study demonstrate that the majority of the preproglucagonexpressing neurons in the NTS project in a target-specific manner to the hypothalamus. It is therefore possible that individual subgroups of GLP-containing neurons can mediate different physiological responses. © 2007 Elsevier B.V. All rights reserved.
1.
Introduction
The nucleus tractus solitarius (NTS) receives visceral afferent information from the cardiovascular, respiratory, gastrointestinal and taste systems in which it plays an important role as both a relay and integrative center (Blessing, 1997). The NTS is topographically organised as different sensory modalities are received and processed at different rostro-caudal levels.
The complexity of the nucleus is highlighted by the numerous neurotransmitters synthesized by neurons in the NTS (Maley, 1996). Over the past decade, the function of a small population of preproglucagon-expressing neurons in the caudal “gastrointestinal” part of the NTS has been scrutinized. The processing of preproglucagon within these neurons is similar to the processing in intestinal L-cells giving rise to mainly glucagonlike peptide-1 (GLP-1) and -2 (GLP-2) and smaller amounts of
⁎ Corresponding author. Rheoscience, Glerupvej 2, 2610 Rødovre, Denmark. Fax: +45 4450 1962. E-mail address: [email protected] (N. Vrang). 0006-8993/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2007.02.043
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glicentin and oxyntomodulin (Bell et al., 1983; Holst et al., 1994; Larsen et al., 1997). Since preproglucagon expression in the brain is limited to the NTS, it has been possible to investigate the projections of these neurons by immunocytochemistry using antibodies raised against preproglucagon or preproglucagon-derived peptides (Jin et al., 1988; Larsen et al., 1997; Rinaman, 1999). These studies have collectively shown that the major target of the brainstem preproglucagon neurons is the hypothalamus. In the hypothalamus, the densest innervation has been observed in two nuclei implicated in the control of food intake and bodyweight, namely the paraventricular (PVN) and dorsomedial (DMH) hypothalamic nuclei. In line with the preproglucagon projection pattern intracerebroventricular (i.c.v.) administration of either GLP-1, GLP-2 or oxyntomodulin has been shown to inhibit food intake in rats (Dakin et al., 2001; Tang-Christensen et al., 1996, 1998, 2000; Turton et al., 1996). Whereas oxyntomodulin presumably acts via the GLP-1 receptor, GLP-2 specifically activates the GLP-2 receptor (Druce and Bloom, 2006; Munroe et al., 1999). Recent studies have revealed an intriguing complexity of the brainstem–hypothalamic preproglucagon system. Whereas the GLP-1 receptor mRNA is expressed in all hypothalamic areas receiving GLP-immunoreactive fibers (Merchenthaler et al., 1999), the GLP-2 receptor expression in the hypothalamus is confined to the compact part of the DMH (Tang-Christensen et al., 2000). The differential distribution of GLP-1 and GLP-2 receptors indeed suggest that GLP-1 and GLP-2 play different roles in appetite regulation despite the fact that the peptides are produced in the same neurons and are derived from the same precursor peptide. To better understand the anatomy of the brainstem–hypothalamic preproglucagon system we employed double retrograde tracing from the PVN and DMH in combination with GLP-2 immunohistochemistry. Using this approach we sought to topographically characterize preproglucagon projections and determine whether the preproglucagon neurons project in a site-specific fashion to the hypothalamus, or whether single GLP-containing neurons target multiple sites.
2.
Results
A total of six rats had injections that were centered in both the PVN and the DMH. The center of the ChB and FG injection sites are shown in Fig. 1. Animals with ChB injections spreading caudally in the DMH or with FG injections spreading rostrally in the PVN were not included in the analysis. As seen in Fig. 1, rats T2140, T2065 and T2114 had ChB injection sites that were centered in the caudal part of the PVN (Plate #27, Swanson, 1998) whereas rat numbers T2126, T2116 and T2138 were centered at the level of the posterior magnocellular PVN (Plate Fig. 1 – Location of ChB and Fluorogold injections in the rats selected for further analysis. A and B are medium power photomicrographs showing the size and location of the PVN (A) and DMH (B) injections (single stained using DAB as chromagen) from rat T2138. The extent and location of the remaining 5 rats have been plotted onto Adobe Illustrator maps from the rat atlas by Swanson (1998). In general, ChB injections are smaller and more clearly demarcated than FG injections (compare A and B). Scale bars in A, B = 100 μm.
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#26, Swanson, 1998). Only animals with FG injections involving the ventral subnucleus of the DMH – that receives the densest GLP-ergic innervation (Larsen et al., 1997) – were included in the analysis. Animals T2140, T2126, T2114 and T2116 were centered in the mid-DMH (Plate # 30, Swanson, 1998), the remaining (T2065, T2138) at a slightly more caudal level (Plate
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#31, Swanson, 1998). In the present study we used a monoclonal mouse antibody raised against human GLP-2 and shown to give rise to the same pattern of immunoreactivity as a previously characterized polyclonal GLP-1 antibody (Larsen et al., 1997). The availability of a monoclonal antibody facilitated the visualization of ChB, FG and GLP-2 in the same sections. Both ChB injections into the PVN and Fluorogold injections into the DMH gave rise to retrogradely labeled cells in the nucleus of the solitary tract. An example of triple stained neuron in the NTS is shown in Fig. 2 and a map of single-, double- and triple-labeled cells in one representative rat is shown in Fig. 3. Cell counts revealed that approximately 25% of ChBlabeled cells in the NTS co-stored GLP-2, whereas 67% of the FG cells co-stored GLP-2, suggesting that the majority of the projection from the NTS to the DMH contain preproglucagonderived peptides. Since preproglucagon-expressing neurons are also found in the reticular formation extending laterally and ventrally from the NTS-proper, cell counts were broken down by region (NTS-proper and “lateral extension”). This part
of the reticular formation contained neurons retrogradely labeled with both ChB and FG and the degree of co-localization with GLP-2-ir was almost identical to that found in the NTSproper (see Table 2). Cell counts were also broken down by rostro-caudal level of the NTS (caudal to the AP, level of the AP and rostral to the AP). These counts revealed that the majority of NTS neurons projecting to the PVN (ChB-labeled) were found at the level of the AP, whereas the majority of the DMHprojection neurons were found in the caudal part of the NTS (see Table 2). As a reflection of the fact that the highest number of GLP-2-positive neurons was found in the caudal part of the visceral NTS (caudal to the AP), this region also contained the highest degree of co-localization between ChB and GLP-2 (45%) and FG and GLP-2 (82%). Furthermore the counting revealed (see Table 1) co-localization between ChB and FG (=neurons projecting to both the PVN and the DMH). Approximately three-fourths of these “two-target” neurons were also GLP-2-positive.
3.
Discussion
This study shows that preproglucagon projections constitute the predominant input from the nucleus of the solitary tract to the dorsomedial hypothalamic nucleus. While approximately 65% of NTS-neurons projecting to the DMH co-stored the preproglucagon-derived peptide GLP-2 only 25% of the NTSneurons projecting to the PVN were found to be GLP-ergic. Furthermore, the study revealed that the majority of the NTSneurons project to either the PVN or the DMH. Only 15–25% of the retrogradely labeled cells were found to project to both hypothalamic nuclei.
3.1.
Fig. 2 – Fluorescence micrographs showing examples of single, double and triple labeled cells in the NTS. A and B are images of the same area but viewed with different emission/ excitation filters. Thin arrow in A and B point to a single neuron labeled with both ChB (red in A), Fluorogold (yellow granula in B) and GLP-2 (blue in B). Large arrow in B points to a neuron containing both FG granula and GLP-2 immunoreactivity. Additionally a couple of single-labeled GLP-2 neurons are seen (arrowheads). Scale bars = 50 μm.
Technical considerations
Clearly, because of the inherent inter-individual differences when performing in vivo neuronal tracing, double-retrograde tracing warrants careful interpretation of the results. In the present study, the two sensitive and well-characterized retrograde tracers cholera toxin, subunit B (ChB) and Fluorogold were used (Lennard et al., 1993; Luppi et al., 1990; Merchenthaler, 1991; Schmued and Fallon, 1986). Because the preproglucagon projections target the PVN and DMH-proper rather than the surroundings, confining the injections to the individual nuclei was not a primary concern, although injections into the DMH extending rostrally into the PVN were excluded from further analysis. Since the GLP-containing nerve fibers course through the periventricular strata on the way to the PVN (Larsen et al., 1997), injections close to the third ventricle at the level of the DMH could potentially damage passing fibers, leading to uptake and false-positive doublelabeled cells. Although the DMH injections inevitably spread into the periventricular strata no injections were centered here (see Fig. 1), making this possibility less likely.
3.2.
Anatomical considerations
A number of studies have examined the projections from the NTS to the hypothalamus using both anterograde and retrograde tracing techniques (Ricardo and Koh, 1978; Sawchenko
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Fig. 3 – Single-, double- and triple-labeled ChB, FG and GLP-2 containing neurons are plotted in single 40-μm tissue sections through the caudal NTS. The distribution of cells in two rats with successful PVN and DMH injections of ChB and Fluorogold, respectively, is shown. The labeling is as follows: GLP-2 (red dots), ChB (open squares), FG (open stars), ChB/GLP-2 co-loc (red squares), FG/GLP-2 co-loc (red stars), ChB/FG co-loc (black triangles), ChB/FG/GLP-2 co-loc (red triangles). The drawings are arranged from rostral (top) to caudal (bottom) and are redrawn from the atlas by Swanson (1998, plates 70 to 73).
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Table 1 – Total and individual cell counts and degree of co-localization between the three antigens examined (cell counts performed in entire visceral NTS and includes the cells extending from the lateral NTS into the RVLM)
# ChB cells # FG cells # GLP-2-ir cells % ChB with GLP-2 % FG with GLP-2 % ChB with FG % FG with ChB % FG/ChB with GLP-2
Total
T2065
T2114
T2116
T2126
T2138
T2140
65 ± 5 45 ± 5 141 ± 17 24 ± 1 67 ± 4 15 ± 1 23 ± 3 72 ± 7
40 39 191 20 72 13 13 100
55 67 173 24 58 22 18 75
68 33 152 21 79 13 27 67
81 38 146 27 68 14 29 64
63 53 89 25 55 16 19 50
86 37 92 28 70 14 32 75
Note that counts were only made in one series of sections out of a total of six. Total counts are expressed as mean ± SEM of the 6 rats included in the study.
and Swanson, 1982; Ter Horst et al., 1989; Thompson and Swanson, 1998). Especially the brainstem projections to the PVN have been extensively scrutinized (Swanson, 1987). Catecholaminergic projections to the PVN have been demonstrated to originate not only in the NTS (A2/C2 region), but also the A1/C1 (caudal ventrolateral medulla) and A6 (locus coeruleus) regions contribute with catecholaminergic fibers to the PVN (Sawchenko and Swanson, 1982). A number of other neurotransmitters found in nerve terminals in the PVN, however, are found in the NTS. Aside from the GLP-1/2containing neurons (Larsen et al., 1997; Rinaman, 1999) also somatostatin-immunoreactive neurons (Sawchenko et al., 1990), cocaine- and amphetamine-regulated transcript (CART) (Fekete et al., 2004) and proopiomelanocortin (POMC) (Kiss et al., 1984) containing neurons in the NTS have been shown to project to the PVN. Our finding that approximately 25% of the neurons projecting to the PVN contain preproglucagon-derived peptides is in line with the complex neurochemistry of this projection. Much less is known about the neurochemistry of NTS projections to the DMH as only a single study has addressed this subject (Sawchenko and Swanson, 1982). In a comprehensive paper on brainstem noradrenergic (and adrenergic) projections to the PVN and SON, the authors note that “in experiments with injections centered still more caudally, in
the dorsomedial nucleus proper, a similar pattern of singleand double-labeling was observed” (Sawchenko and Swanson, 1982). The authors found dopamine-beta-hydroxylase immunoreactivity in approximately 75–80% of true-blue containing neurons in the NTS (A2–C2 region) following PVN injections, and apparently (although not quantified) the same pattern was observed in cases accidentally involving the DMH (Sawchenko and Swanson, 1982). Although we have not examined the degree of co-localization between Fluorogold and catecholamine neuron markers our finding of a high degree of co-localization (approximately 65%) between Fluorogold and GLP-2-ir, suggests that catecholaminergic input from the NTS to the DMH constitute less than 35% of this connection, since the preproglucagon neuronal population is separate from the catecholaminergic neuronal pool in the C2/ A2 region (Larsen et al., 1997). In the present study, GLP-2-positive cells projecting to the PVN and DMH were found in both the NTS proper as well as in the wing of GLP-2-positive neurons extending laterally through the dorsal reticular area into the A1 area (at the level of the caudal part of the area postrema). The fact that similar degrees of co-localization were found in the NTSproper and the “lateral extension” indeed suggests that the pool of GLP-2 within and outside the NTS constitutes a functional homogeneous cell group rather than two separate
Table 2 – Cell counts and degree of co-localization divided into different anatomical sub regions
# ChB cells # FG cells # GLP-2-ir cells % ChB with GLP-2 % FG with GLP-2 % ChB with FG % FG with ChB % FG/ChB with GLP-2 # ChB with GLP-2 # FG with GLP-2 # ChB with FG # FG/ChB with GLP-2
Total
NTS proper
Lateral extension
Caudal NTS
NTS-AP-level
NTS-rostral to AP
65 ± 5 45 ± 5 141 ± 17 24 ± 1 67 ± 4 15 ± 1 23 ± 3 72 ± 7 16 ± 2 29 ± 2 10 ± 1 7±1
48 ± 7 34 ± 4 106 ± 11 25 ± 2 66 ± 5 16 ± 3 22 ± 4 66 ± 11 12 ± 1 21 ± 1 7±1 5±1
18 ± 6 11 ± 3 34 ± 8 24 ± 4 82 ± 9 21 ± 10 46 ± 18 72 ± 16 5±1 8±1 3±1 2±1
27 ± 3 28 ± 3 114 ± 17 45 ± 5 84 ± 4 25 ± 3 25 ± 3 79 ± 7 12 ± 1 23 ± 3 7±1 6±1
31 ± 6 14 ± 3 27 ± 5 11 ± 3 50 ± 12 8±3 25 ± 7 58 ± 19 4±3 6±2 3±1 2±1
10 ± 2 3±1 0 0 0 0 0 0 0 0 0 0
Number of labeled cells counted in the entire visceral NTS (total numbers include counts at all rostro-caudal levels and include cells located in the lateral extension of the NTS). These cell counts have been broken down into: 1) NTS-proper and the lateral extension of the NTS and 2) different rostrocaudal levels (caudal to the AP, at the level of the AP and rostral to the AP). Note that counts were only made in one series of sections out of a total of six. Total counts are expressed as mean ± SEM of the 6 rats included in the study.
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populations of neurons as would be predicted from topographic criteria. This is in line with previous data from our laboratory showing c-Fos induction following gastric distension in GLP-2-immunopositive neurons located both in the NTS proper and in the ventrolateral medulla (Vrang et al., 2003).
3.3.
Fig. 4 – Laser confocal images (5 μm thick optical sections) showing (A) GLP-1 and (B) GLP-2 immunoreactivity in the NTS. GLP-1-ir was visualized using a rabbit–polyclonal antibody (#2135) whereas GLP-2-ir was visualized using a mouse monoclonal antibody (#F1221). As seen in C the two peptides co-localize 100% in both neuronal and axonal-like structures. Scale bars = 50 μm.
Functional considerations
A number of studies have examined the possible functional role of the brainstem preproglucagon-expressing neurons. Based on the anatomy of the central preproglucagon system, we and others hypothesized a possible role of preproglucagonderived peptides in appetite regulation. Since the initial studies reporting anorectic effects of centrally administered GLP-1 (Tang-Christensen et al., 1996; Turton et al., 1996), a number of studies have demonstrated that indeed GLP-1 reduces food intake — an effect that is mediated by the GLP-1 receptor as GLP-1 receptor antagonists can block the effects (see Larsen et al., 2003). In contrast to the wide hypothalamic and brainstem distribution of GLP-1 receptors, the GLP-2 receptor expression is much more limited. In the hypothalamus the expression is limited to the DMH, but also few extra hypothalamic sites contain GLP-2 mRNA albeit at lower levels (hippocampus, premamillary nucleus, substantia nigra, lateral parabrachial nucleus) (Lovshin et al., 2004). The short-lasting anorectic effects of centrally administered GLP-2 is presumably mediated by the GLP-2 receptor-bearing neurons in the DMH, which was also the only hypothalamic area that contained a significantly higher number of c-Fos-ir cells following GLP-2 administration (Tang-Christensen et al., 2000). These data suggest that by activating DMH neurons a short-term reduction in food intake can take place. The DMH projects densely to parvocellular parts of the PVN that could be responsible for mediating the observed anorexia (Thompson et al., 1996). While it is generally accepted that the PVN plays a crucial role in regulating a variety of homeostatic functions (e.g. appetite, thirst, stress, temperature) the role played by the DMH in hypothalamic integration is somewhat controversial. A large number of lesion studies support the notion that the DMH plays a role in controlling the bodyweight set-point, in that animals with DMH lesions are capable of regulating their bodyweight, albeit at a lower level (Bernardis and Bellinger, 1998). Although interpretation of lesion studies is hampered by the complexity of the DMH, recently other data have accumulated that point to a role of the DMH in regulating energy balance. First, the DMH contains a population of PVNprojecting neuropeptide Y (NPY) mRNA-expressing neurons that are activated in lactating rats, i.e. at a time of increased body-energy demand (Li et al., 1998a,b). Second, NPY mRNA expression is increased in the DMH in the hyperphagic OLETF also pointing to a role of these neurons in animals with an increased drive to eat (Bi et al., 2001). Our present anatomical findings do not readily answer what differences might exist between GLP-1- and GLP-2mediated anorexia, but the fact that the preproglucagon neurons only have partly overlapping projection systems suggests that separate populations of preproglucagon neurons could be involved in different aspects of feeding behavior. The results presented here suggest that even within this relatively
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small group of neurons, separate populations with anatomically diverse projections exist highlighting the necessity of careful anatomical analyses as the basis to understand brain physiology.
4.
Experimental procedures
4.1.
Animals and surgery
Thirty-five male Wistar rats were used for the in vivo neuronal tracing experiments. The rats were kept on a 12-h light, 12-h dark cycle (lights on at 0600 h) in a temperature-controlled environment (22–24 °C) with free access to food (Altromin standard chow 1243; C. Petersen, Ringsted, Denmark) and water. All experiments were conducted in accordance with internationally accepted principles for the care and use of laboratory animals and were approved by the Danish Committee of Animal Research. Rats were deeply anaesthetised using hypnorm dormicum (1.0 ml/kg; containing 0.08 mg fentanyl, 2.5 mg fluanisone and 5.0 mg medazolam per ml) and placed in a Kopf stereotaxic apparatus. The incisor bar was set 2 mm below the ear bars. A glass electrode with a tip diameter less than 25 μm was filled with 4 μl of a cholera toxin, subunit B (ChB; choleragenoid, Sigma Chemicals Co., MO, USA) (1% w/v diluted in phosphate-buffered saline containing 50 mM potassium, KPBS) and another with Fluorogold (2% diluted in 0.9% NaCl). Both ChB and FG were injected iontophoretically using a positive current of 10 μA (7 s on, 7 s off) for 10 min, then left in situ for another 10 min to avoid spreading of tracer upon removal. Injection co-ordinates for ChB injections into the PVN were: antero-posterior = −1.8 mm (relative to bregma), lateral = 0.5 mm, and 7.6 mm below the dura. Injection co-ordinates for FG injections into the DMH were: antero-posterior = − 2.6 mm (relative to bregma), lateral = 0.5 mm, and 8.4 mm below the dura. At 7 days postoperatively, the animals were reanaesthetized and vascularly perfused with heparinized KPBS (15,000 IU/l), followed by 4% paraformaldehyde dissolved in 0.1 M phosphate buffer (pH = 7.4) for 15 min. The brains were removed and post-fixed in the same fixative overnight – then stored in KPBS until further processing. The brains were cryoprotected for two days in a 30% sucrose–KPBS solution and one-in-six series of 40-μm-thick frontal sections were cut (fore- and hindbrain separately) on a freezing microtome and collected in cryopreservant (Watson et al., 1986) and stored at −20 °C. All reactions were carried out on free-floating sections.
4.2.
Immunohistochemistry
4.2.1.
Single immunohistochemistry
To determine the precise injection sites one forebrain series from each rat was immunoreacted with an anti-FG antibody (rabbit anti-Fluorogold, Fluorochrome Inc., Englewood, CO) and one series with an anti-ChB antibody (goat anti-ChB; LIST Biologicals, Campbell, CA). Both antibodies were diluted 1:4000 and prepared in KPBS with 0.3% Triton X-100 and 1.0% bovine serum albumin (BSA). The sections were stained using the indirect avidin–biotin method and diaminobenzidine as a chromagen as described in detail previously (Vrang et al., 1995).
4.2.2. Double staining to determine the overlap of GLP-1 and GLP-2 (characterization of monoclonal GLP-2 antibody) While the monoclonal GLP-2 antibody used in the present study has been raised against full-length human GLP-2 (GLP21–33) and previously shown to stain neuronal cell bodies overlapping exactly with preproglucagon mRNA-expressing neurons in the NTS, the degree to which the GLP-2 immunoreactivity overlaps with GLP-1 immunoreactivity has not been examined (Tang-Christensen et al., 2000). In order to examine the degree of overlap between GLP-1-ir and GLP-2-ir in the brainstem, we used a double immunohistochemical detection procedure. GLP-1 immunoreactivity was detected using a GLP1 antibody (#2135; Larsen et al., 1997) and the double staining was performed according to the procedure below. As seen in Fig. 4, we found 100% co-localization between the two antibodies. These data support previous results demonstrating that preproglucagon in the NTS is processed to yield GLP-1, GLP-2 and oxyntomodulin (Larsen et al., 1997).
4.2.3.
GLP-1 and GLP-2 double staining procedure
Brainstem test sections from male Wistar rats perfused transcardially with 4% paraformaldehyde were reacted as follows: First, the sections were washed 3× for 10 min each in KPBS, then for 10 min in 0.1% H2O2 in KPBS followed by 20 min in 5% swine serum in KPBS with 0.3% TX and 1.0% BSA (KBPS– BT). After 3× for 10 min each in KPBS, the sections were incubated in the primary antibodies: mouse anti-GLP-2 diluted 1:200 (F12-21, generously provided by Jes T. Clausen, Novonordisk A/S; complete overlap with preproglucagon mRNA shown in Tang-Christensen et al., 2000) and rabbit anti-GLP-1 diluted 1:5000 (#2135, characterized in Larsen et al., 1997) in KPBS–BT. The next day, sections were rinsed 3× for 10 min each in KPBS containing 0.1% TX and 0.25% BSA (KPBS-T) then incubated in biotinylated donkey anti-rabbit 1:1000 (Jackson Immuno Research Laboratories) diluted in KPBS–BT. After 3 rinses in KPBS-T, 60 min in ABC–streptavidin horseradish peroxidase (Vector Elite Kit™; Vector Laboratories, Burlingame, CA) and 3 washes in KPBS-T, sections were reacted for 12 min in biotinylated tyramide (TSA; biotinylated according to the procedure described by Adams (1992) using tyramine–HCl (Sigma Aldrich) and sulfosuccinimidyl-6-(biotinimide) hexanoate (NHS–LC–biotin; Pierce, Rockford, IL)) diluted 1:100 in KPBS-T containing 0.005% H2O2. Sections were rinsed 3 times in KPBS-T incubated for 60 min in streptavidin Alexa 546 (Molecular Probes, Eugene, OR) diluted 1:200 in KPBS-T. Next, sections were rinsed 3× for 5 min each in KPBS-T and subsequently incubated for 10 min in 1% H2O2 in KPBS (30 ml 35% H2O2/ml) to block the added peroxidase. Following 3 rinses for 10 min each in KPBS-T, sections were incubated for 60 min in peroxidase-coupled secondary antibody (HRP-coupled antimouse antibody diluted 1:100 in KPBS-T; Jackson Immuno Research Laboratories). After 3 rinses in KPBS-T, sections were incubated for 12 min in fluorescein-labeled tyramine (FITC– TSA; labeled according to the procedure described by Adams (1992) using tyramine–HCl (Sigma Aldrich) and sulfosuccinimidyl-6-(fluorescein) hexanoate (NHS–LC–biotin; Pierce, Rockford, IL)). After this reaction, sections were rinsed 2× for 10 min each in KPBS-T and finally for 10 min in distilled water. Slides were dehydrated through a graded series of ethanol dilutions (30%, 60%, 80% 96% 99% and xylene) cover slipped in Pertex™.
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4.3.
Triple staining – GLP-2, ChB and FG co-localization
GLP-2, ChB and FG were visualized simultaneously by a double immunohistochemical detection procedure and direct visualization of FG using a UV-filter on the fluorescence microscope. Brainstem sections from rats with successful PVN and DMH injections were reacted as follows. Firstly, the sections were washed 3× for 10 min each in KPBS, then 10 min in 0.1% H2O2 in KPBS followed by a 20-min incubation in 5% swine serum in KPBS with 0.3% TX and 1.0% BSA. After 3 rinses for 10 min each in KPBS, the sections were incubated in the primary antibodies: mouse anti-GLP-2 diluted 1:6000 (F12-21, generously provided by Jes T. Clausen, Novonordisk A/S) and goat anti-ChB diluted 1:500 in KPBS–BT. The next day sections were rinsed 3× for 10 min each in KPBS–BT before incubation in biotinylated donkey anti-mouse 1:2000 (Jackson Immuno Research Laboratories). After 3 rinses for 10 min each in KPBST followed by 60 min in ABC–streptavidin horseradish peroxidase the sections were washed 3× for 10 min each in KPBS-T. Sections were then reacted for 12 min in biotinylated tyramide diluted 1:100 in KPBS-T containing 0.005% H2O2, rinsed 3× for 5 min each in KPBS-T and incubated for 60 min in Streptavidin Alexa 350 (Molecular Probes) diluted 1:200 in KPBS-T. Following 3 rinses for 5 min each in KPBS-T, the ChB antibody was detected by incubating the sections for 60 min with a Texas-Red-labeled anti-goat antibody diluted 1:100 in KPBS-T (Jackson Immuno Research Laboratories). After this reaction, sections were rinsed 2× for 10 min each in KPBS-T then dehydrated through a graded series of ethanol dilutions (30%, 60%, 80% 96% 99% and xylene) and cover slipped in Pertex™.
4.4.
Photomicrographs
Sections double-labeled with GLP-1 and GLP-2 were examined using a Zeiss LSM510 laser confocal microscope and images (5 μm thick optical sections) acquired using LSM510 software. Images of triple labeled (GLP-2, FG and CHB) cells were acquired using a Nikon ACT-1200 digital camera mounted on a Nikon E1000M microscope. Single, double and triple labeled cells were scored in one series of triplelabeled sections (brainstem cut in 6 series, hence sections were spaced 240 μm apart). All sections from this triplelabeled series through the NTS were scored for GLP-2, FG, ChB single-, double- and triple-labeled cells by an observer blinded to the different treatment groups. The AP was used to define three rostro-caudal levels of the visceral NTS: caudal to the AP, AP (sections containing the AP) and rostral to the AP. In addition the counts were divided into NTS and lateral extension of the NTS (lateral reticular formation that contains neurons projecting to the PVN and DMH as well as a group of GLP-positive neurons). Although GLP-2 and Fluorogold were visualized using the same filter settings, the grainy yellow Fluorogold contrasted clearly with the homogenous blue color emitted by the Alexa-350 dye (see Fig. 2). All images were enhanced for brightness and contrast in Adobe Photoshop 7.0 and combined into plates using Adobe Illustrator 10.0. Adobe Illustrator plates (Plates 70 to 73) from the atlas by Swanson (1998) were used to generate the map of labeled cells in Fig. 3.
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Acknowledgments This study was supported by The Danish Diabetes Association, The Novo Nordisk Foundation and Fonden til Lægevidenskabens Fremme. We thank Susi Jensen, Anja Daniel Andersen and Farida Sahebzadeh for excellent technical assistance.
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